Plant Physiol.

(1969) 44, 886-892

Enzymic Degradation of Starch Granules in the Cotyledons of

Germinating Peas' 2
Bienvenido 0. Juliano3 and J. E. Varner
AEC Plant Research Laboratorv, Michigan State University, East Lansing, Michigan 48823
Received January 30, 1969.

Abstract. Starch, total a- and f3oamylase, and phosphorylase levels and the zymogram
patterns of these 3 starch-degrading enzymes were determined in the cotyledons of smooth pea
(Pisum sativum L.) during the first 15 days of germination. Starch is degraded slowly in the
first 6 days; during this time, a-amylase is very low, $-amylase is present at ?a constant level
while phosphorylase gradually increases and reaches a peak on the fifth day. Beginning on
the sixth day there is a more rapid degradation of starch which coincides with a-amylase
production. One phosphorylase band and 2 /-amylase bands are present in the zymogram
of the imbibed cotyledon. An additional phosphorylase band and 1 a--amylase band appear
during germination. Seeds imbibed in benzyladenine, chloramphenicol, and in cycloheximide
show retarded growth and slower starch degradation and enzyme production than the controls.
We conclude that a-amylase is the major enzyme involved in the initial degradation of starch
into more soluble forms while phosphorylase and l3-amylase assist in the further conversion
to free sugars.
The enzynmic degradation of starch in plants has
been attributed to phosphorylase or a-amylase in the Materials and Methods
leaves (6, 9) and to a-amylase in starch storage
tissuies (30) on the basis that starch degradation Pea seeds (Pisumi satizvum L.) cv. "Early Alaska"
usually coincides with periods of maximum activity from the Farm Bureau Services, Incorporated,
of these enzymes. In an effort to determine nmore Lansing, Michigan were soaked for 5 to 6 hr in
directly and more precisely the relative importance distilled water, dipped for 5 min in 1 % sodium
of these enzymes in the breakdown of reserve starch hypochlorite, washed with sterile water and planted
granules, the cotyledons of germinating peas were on moist vermiculite in the diffuse light of the
examined because changes in the levels of amylase laboratory at 24 to 26°. Pretreatment with various
(30, 35), starch ( 1), amvlase and starch (3, 29), metabolic regulators involved soaking the seeds for
and phosphorylase (31) have already been- much 24 hr in aqueous solutions of benzyladenine, chloram-
studied in this tissue in different varieties and under phenicol, or cycloheximide.
different germination conditions. In our study we For carbohydrates determination, 1 or 2 cotvle-
have followed the levels of starch, dextrin, free dons were excised from seedlings, rinsed witlh water,
sugars, amylases, and phosphorylases of the cotyle- blotted. and soaked in hot 80 % (v/vr) ethanol and
dons of the cultivar "Early Alaska", a variety of ground in Pyrex glass tissue grinders. The-homog-
smooth seeds, during germination in the light. The enate was centrifuged for 10 min at 20,OOOg and the
starch granules remaining in the cotyledonary cells supernatant fluid collected as the free sugars fraction.
after various times of germination were also char- The residue was then extracted twice with cold
acterized with respect to their physical and chemical 5'0 % perchloric acid and centrifuged each time for
l)roperties to obtain additional information about the 20 min at 25,000g. The combined extract contained
degradation which had already occurred. the starch and dextrin. For separate dextrin ex-
tractioni, cold water extraction of the residue from
ethanol extraction was performed beforel that of
perchloric acid. Carbohydrate concentration of the
Work carried out under United States Atomic EnI- extracts was determined colorimetrically by the
ergy ComImiissioIn contract No. AT( 11-1)-1338 and a anthrone-sulfuric acid nmethod of McCready et al.
research fellowship (B. 0. Juliano) from the Interna- ( 19) with glucose as standard, aind results were
tional Rice Research Institute. expressed as mg glucose/2 cotyledons.
2 We acknowledge the assistance of Dr. J. G. Scan-
dalios and E. S. Chao on polyacrylamide gel electro- Protein extracts were prepared by cutting pre-
phoresis techniques. viously rinsed and blotted cotyledons (2) to, small
3 Present address: Chemistry Department, The Inter- pieces and homogenizing in 2 ml of cold 0.05 M
national Rice Research Institute, Los Banos, Laguna, pH 7.0 tris buffer in a Pyrex glass tissue grinder
The Philippines. (ice bath). The resulting mixture was centrifuged
886
 JULIANO AND VARNER---STARCH DEGRADATION IN GERMINATING PEAS 887
for 10 min at 20,OOOg and the supernatant liquid 0.003 % calciuim chloride and 0.006 % KHJ'Otwas
analvzed wvithout further purification for protein determined at room temperatture in the presence of
content (bovine serum allbumini standard) according toluiene vapor.
to Lowry ct al. (18), for total amylase activity by- Starch granules were isolated from cotyledons
the method of Chrispeels and 'Varner (4), for phos- of 0 day (dry seeds), 5-day. 9-dav, and 11-day
phorvlase activity as described by Lee (16), and an seedlings b1 the metlhod of Greenwood and Thomson
aliquot used for polyacrylamide gel electrophoresis (13). The excised cotvledons w ere soaked over-
(27, 28). MIodifications of the amvlase assay in- night in 0.01 WI ercuiric chlloride, w\ashed witlh wvatel-.
cluded the use of instead of 1.0 nml iodine solution and homiogenlize(d in cold 4 % NaCl in a WNaring
and 4.5 instead of 5.0 ml water due to the presence Blendor at mledituni speed for 3 lin. The hrei was
of iodine-reducing substances in the extract. and the filtered througlh cheeseclotlh, shlaken with tolutelne for
use of O.05 M pH 4.8 acetate buffer instead of phos- -at least 6 hr. an(d the toluienie layer with deenatulred
pliate buffer to stippre-s phosphorylase activity dtur- l)rotein siphoned off. Extraction was repeated 3
ing the assay. Total (a- anid P-) amylase activity times or uintil the toluiene laver was n1o longer tuirbid.
was expressed as the (lecrease in absorbance at The starch suispension was theni centrifuiged 15I mill
620 mniu per niiii pel 2 cotyledons. The phosphorylase at .500g. suis-pelnded thrice in water. recentrifulged.
assav was d(one at roomii temperature an(l activity anid air-dried at room temlperature. Recovery of the
was calculated as uinloles Pi liberated per min per starch ranged froml 45 to 70 % Mean graniule size
cotvledons. of the samples was obtained hy measulr-ing the major
Polvacrvlamide gel electrophoresis followed a axis of the grantule from photomlicrogrraphls miagniified
modification of the procedure of Scandalios (27) 400-fold.
using a 9 :1 (v/v) mixture of 0 8 mm citric acid- Starch was defatted with refltuxinig 85 % (v'/v)
0.04 -m tris pH 8.2 bbuffer and 0.06 Am lithium h- methanol for 24 hr. air-dried, pre-gelatinized with
droxide-0.019 M boric acid pH 8.9 btuffer in the ,gel, hot dimethvl sulfoxide. and fractionated as previously
and the latter buffer for the reservoirs. An 8 %/ described (2). The resulting fractions were ania-
gel was employed to avoid molecular exclusion of lyzed for 8-amvlolvsis limits using Worthington
enzymes at the origin. The electrophoresis was run Biochemicals Corporation crystalline /3-amvlase (13),
for 5 to 6 hr at 40 with a current of 2.5 milli- blue value (absorbance at 680 m1l) (10), intrinsic
amperes/cm. The gels were then incubated at am- viscosity in 1 x K-OH at 300 using No. 50 or 100
bient tenmperature for at least 4 hr in 0.07 M pH 5.7 Ubbelohde dilution viscometers (11), and amvlo-
phosphate buffer wvith 0.8 % (wv/v) MIerck soluble pectin mean chain length by periodate oxidation (13).
starch as substrate to assay for phosphor-lase and One-tenth Mc of a-D-glucose-U-14C (Schwarz
amylases, in 0.1 AI pH 5.1 citrate buffer containing Bioresearch, Incorporated) or sucrose-U-14C (Nu-
0.7 % (w/v) glucose-I-P (dipotassium salt dihvdrate, clear Research Chemicals) w as applied to the blotted
Nutritional Biocheniicals Corporation) and 0.8 % inner surface of a cotyledon of pea seedling. After
(w/v) oyster glvcogen as primer to assay for phos- 20 to 24 hr assimilation time, the cotyledons were
phorylase or with 0.8 % (w/v) solutble starch in washed thoroughly witlh water and homogenized in
'0.05 m tris-maleate buffer pH 5.6 for amylases. hot 80 % ethanol. Starch was isolated according to
After this, the gels were stained with dilute L-KI Hassid and Neufeld (15) without pregelatinization.
solution for a few min to show the zymogram bands. and the activity of the starting brei and the starch-
and fixed for several hr in 5 % (v/v) acetic acid in iodine complex was determined from an aliquot w\vith
30 % (v/v) methanol (28). a Nuclear Chicago Model 1043/1044 planchet counter.
Crulde extracts from dry or imbibed cotyledons,
anid from cotyledons of 3 to 10 day old seedlings
were obtained by homogenizing them in a minimum
Results
quantity of tris buffer in a mortar and pestle and
centrifuging the mixture 10 min at 20,000g. Frac- Kinetics of Starch Degradationi anid of Phos-
tionation of this extract was studied at various phorylase and Amnylase Activity ini Cotyledons of
ammonium sulfate concentrations of 20, 30. 40, 50. Germinating Peas. The degradation of starch in
60, and 70 g/ml extract and zymograms made of germinating pea cotyledons consists of a slow phase
both precipitate and supernatant fractions. The during the first 5 to 6 days of germination in which
seedling extract was fractionated by cold ethanol starch content decreases by 25 % and a fast stage
addition to ethanol concentrations of 40, 45, 50, 55., in which the remaining starch is degraded up to
and 60 % (v/v) followed by electrophoresis of the 5 times as fast as during the slow stage (Fig. 1).
supernatant fractions. These amylase preparations The residual starch after 15 days germination is
were identified polarimetrically according to Robyt only 1 % of the -original starch content. The original
and French i(25) using 0.1 % pea amylose in 0.05 M starch content of the cotyledon after correction for
pH 4.8 acetate buffer as substrate, and a Perkin dextrin content is 98 mg glucose or 88 mg anhydro-
Elmer Model 141 polarimeter. The action of these glucose/2 cotyledons. This value corresponds to
extracts on a suspension of Remazolbrilliant blue 45 % of the dry weight, based on a dry weight of
R-starch (24) (Calbiochem Amylose Azure) in 194 mg for 2 cotyledons. Reported valtues for smooth
g88 PLAN'r PHYSIOLOGY
The level of protein in the cotyledon extracted in
tris buffer decreases slowly during the first 5 days
of germination. Thereafter it decreases rapidly and
is only 7 % of the original content hby the fifteentlh
day (Fig. 1). In some cases, the protein contenit
of the fifth day extract was higher than that of the
third dav. This l)rotein fractioin represents 50 % of
the crudcle protein content in the inmbibed seed alnd
lrobably contains sonme decreases
globulin as vvell as the
albumnis (5). Similar in protein level
have been reported bv others (1, 3, 5). Amylase
level in the tris extract riemains low and constant
(luring the first 4 days of gernminatioin and increases
to a maximunm (6-fold) level by the ninth dav. This
period coincides with the rapid decrease in extracted
protein. Althouigh this colorimiietric assay has beeni
used predominantly for a-amvlatse activitv, fl-amylase
zalso reduces the iodine-blue color of starch at a
< -, 5 1-01-5 - slower rate than a-amvlase. Simlilar increases in
DAYS GERMINATED
FIG. 1 Changes in the level of starch, dextrin, free
aictivity of amylase during this period have been
sugars, extracted protein, total amylase, and phosphory-
reported for this cultivar groNvn in the light (33)
lase in the germinating pea cotyledons. and in the dark (35).
Phosphorylase activity increases, to a 3-fold level,
bv the fifth day of germinatioin. Swain and Dekker
pea seeds including testa aInd axis tissues ranged (31), usinig the same cuiltivar grown in the dark,
from 42 to 45 % (13,19). The other carbohydrate noted an increase in phosphorylase activity o,f more
fractions, dextrin and free sugars, do not show a than 13-fold between the first and fouirth day of
peak but decrease gradually from the 0 day value. ,germination.
The starch degradation of this cultivar occurs earlier
and faster than that reporte(l for other v.arieties The Pattern of Starclc-degradinqg Enzymiies int tlle
(1,3. 29). Pea Cot vledons. The zvmogranii )attern of the
starch degrading enzynes sos a maxilliill of 6
hands ( Fig. 2). The 2 slowest hands acnd the
DAYS GERMINATED fastest band were colorless in gels incubated in starch
O O 3 in phosphlate buffer and stained wi.h L. hut only the
5 9 9 9 fastest band showed activity with the samle substrate
in tris-maleate bulffer. The 3 mid(lle bands were
pink in color after incubation in starchl anlid staining
with. L, and were present in starch-inctubated gels
SLOW P regardless of the buffer used. The 2 slow batndls
were the only ones which svyntlhesize(d blue-iodline-
FAST P staining bands froml glycogen plrimer acnd glucose-l-1l.
These results are consistent with the identification
s - = . SLOW 1-A
of these 2 slowest bands as phosphorvlases. T'he
canl degrade starch onlI' in the presence of P1; this
r. INTER. a-A explains their ;absence in the gel incubated in tris-
FAST -A mlaleate buffer. Longer incubation tinme was required
when glvcogeni wvas deleted fromii the citrate buffer
containing glucose-l-P. Although this nmay mean
that the phosphorylases do not re(uire a primer as
does the imiaize phosphorylase II of Tsai and Nelsoni
(32), a inore likely explanation is that the phos-
A
phorylase has sonme lprimer complexed witlh it. Stuch
+ B C phosphorvlase-glycogen comlplexes were observed inl
FIG. 2. Schematic zymogram of germinating pea gels in which 0.2 % glycogen was added to the gel
cotyledon a- and f-amylases (A) and phosphorylases before polymerization. The portion incubated in
(P). O' denotes 0-day extract stored 5 days at 4°. In- glucose-l-P showed, on iodine staining, 3 bands of
cubation medium: 0.8 % (w/v) starch in phosphate buf- which only the slowest one showed activity with
fer (A) ; 0.7 % glucose-l-P and 0.8 % (w/v) glycogen starch in phosphate buffer as substrate. No addi-
as primer in citrate buffer (B); or 0.8. % (w/v) starch tional phosphorylase band appeared with the additioni
in tris-maleate buffer (C). Stain was iodine. of 0.1 % AMP to the incubation medium indicating
 JUL'.IANO AND) VARNER-STARCIf DEGRAI)ATION IN GER-MINATING PEAS (R(c9
the absence in cotyledons of germinating pea of hydrolysates from the action of this enzyme on amy-

phosphorylases which require this coenzyme (7). lose showed a downward mutarotation. These are
characteristics of a-amylase. The enzyme slowly
The pink color reaction of the bands with inter- hydrolyzed Remazolbrilliant Blue R-starch into a
mediate migration rates with starch and amylopectin deep-wblue solution. Only a-amylase can liberate a
indicate incomplete hydrolysis of the substrate. blue dextrin-dve hy-drolysate from this starch de-
These bands were absent in gels incubated in rivative. The blue dye is covalently linked to the
glvcogen plus glucose-i-P. Fractionation with am- primary hydroxvl group of starchi and this dye
mlonium sulfate of extracts of imbibed cotyledons suibstituent is a barrier to /3-amylase and phos-
showed that most of the activity of this enzyme was phorylase action on starch ( 17, 24). These reactions
precipitated with 20 g ammoniumn sulfate/100 ml permit identification of this band as a-amylase.
extract as evident from the zymograms. Polari- Thus, of the 6 starch-degrading enzymes of cotyle-
metric study of the action of this enzyme preparation dons of germinating smooth pea, 2 are phosphorv-
on amvlose in acetate buffer g(ave an ul)ward mutaro- lases, 3 are /3-amylases and 1 an a-amylase.
tation of the hydrolysate on sodium carbonate addi- Changes of the Enzyme Patternt During Germi-
tion. This indicates that the sugar hvdrolysate had nation. In the 5-hr imbibed seed, only the slow
the fl-configuration (25). This extract has no ac- phosphorylase and the /8-amylase bands were detected.
tivity toward Remazolbrilliant Blue R-starch, but The fast phosphorylase and the a-amylase bands are
starch can be hydrolyzed by a-amylase but not by detectable by the third day of germination. The
hydrolyzes amylopectin. Remazol'brilliant Blue R- fast /3-amylase band appeared about the ninth day of
fl-amylase (24). Heating for 15 min at 700 inacti- germination but it also appeared during 0° storage
vated this enzyme, which is typical for 83-amylase of an extract from imbibed seeds (Fig. 2). Thus,
(8). These properties formed the bases for desig- the fast phosphorylase and the single a-amylase
nating these enzyme bands as /3-amylase. /8-Amylase bands are presumably the only 2 new enzymes in the
has been shown to be present in the mature pea cotyledons which are produced during germination.
cotyledon by Swain and Dekker (31) but their In gels with 0.2 % amylopectin, a blue-staining band
,8-amylase from axis tissue was more soluble in vas observed in germinated seedlin,g extracts with
ammonium sulfate than this cotyledon enzyme. In a mobility intermediate between the phosphorvlases
fact, the cotyledon fraction precipitating between the and ,3-amylases. This probably is the R or de-
salt concentrations of 23 to 53 g/100 ml had no branching enzyme which can synthesize amylose from
/3-amvlase bands in its zymogram. Although Q amylopectin and which has been detected electro-
(branching) enzyme also has the same color reaction phoretically in maize endosperm (20).
with starch as /3-amylase. the former has little Properties of Starch in the Pea Cotyledons.
activity on amylopectin (14), whereas /8-amylase is Some properties of the starch granules isolated from
active on this substrate. the mature seed and germinating pea cotyledons are
The fastest migrating band in the zymogram from shown in table I. Most of the granules had cracks
pea cotyledons was more soluble in ethanol than along their major axis. The values obtained for the
/8-amylase since no
fl-amylase bands were noted in seed (0-day) starch were comparable to previously
the 50 % ethanol supernatant liquid upon assay. reported values for smooth pea starch for granule
This enzyme was stable to heating for 15 min at size of 30 to 40 /A (13, 23); /8-amylolysis limits of
700 (8) at pH 5.3 in the presence of Ca2' and 80 and 81 % (13): amvlopectin intrinsic viscosity

Table I. Somne Properties of Cotyledon Starch Granutles of Germ7linatintg Peo

Days germinated
Property 0 5 11
Starch
Mean granule size, , 28.9 + 2.2 25.0 + 1.2 20.8 +- 1.4
Blue value, Abs. at 680 mju 0.351 0.368 0 366
Amylopectin
Intrinsic viscosity, nil,'g 182 112 95
Mean chain length, anhydro-glucose units 26.6 + 0.9 26.5 -e(0.6 21.8 -+ 0.0
/3-Amylolysis limit, % 57 53 47
Apparent iinner chain length,'
anhydro glucose units 9 10 9
Amylose
Blue value, Abs. at 680 m, 1.02 0.92 1.05
Intrinsic viscosity, ml/g 170 163 151
/3-Amylolysis limit, % 84 89 96
Calculated from chain length-[(chain length X /-limit) - 2.5]: results were corrected to nearest whole num-
ber (13).
890" PLANT PHYSIOLOGY

of 140 to 15/7 mil/g (13, 23); of 57 and 58 % (13); a

Z I rrl_
n-limiit and mean chain len,gth of 26 to 27 anhvdro-
glucose utnits ( 13, 23). -J
w
The miean grantile size of the starch decreases
dtiring germiniiiation (table 1). A slight increase in
the blue value dutrinig germination indicated that the
-
0
U)
N
--a---*-- AA

amvilopectinl fr,action was sliglltly- mlor-e suisceptible 0

iO _\~
5C0
to enzynnatic solibilizatioll in tile starcil grantile thani -

0
was anmviose. 0
8 * H20O
The nmolectu-lar size of botlh starch fractions as I
30 f./ml BENZYLADENIN\
U
indexed 1w) intrinsic viscosity decreased (du ring ger- I-
A 2 mg/m CHdLORAMIPHENICL
minationi. However, the degradation of tlle anlvlo- V 10,u.g/ml CYCLOHEXIMID \
pectin fraction was illucil miore extensive 1y the fifth
day, especially considering that a brlanclled plolvymler
like amnvlopectin chlaniges less in viscosity per unit N
z
4
4O o-f
4;I4
4L4C
1 1 11
cliailge in ilolectilar size tilali its linear analogue,
amvlose. Since its chain length remlainedl tile same
tip to this tinme, this dirol) in viscosity miust be dute to 4
scissioIl of inner chiaini a-l,4-glucosidic linkages by 2
_
.-A
........
-t'
2
a-amn lase. The amvlopectin of the residtual starch (n
of the eleventh day sallple lla(l a silorter outter chain -J
length tllan the earlier samples, probably becaause of 4 v Jl
the actioil of 8-amylase alnd of phosphorylase. Such 5 10 14
increases in fl-limit values can be explained only by DAYS GERMINATED
ca-amylolysis of inner a-1,4-glucosidic bonds, which FIG. 3. Changes ill the starch and total amylase con-
exposes more non-reducing ends to ,8-anvlvase action. tents of germiinatinig pea cotyledons pretreated witlh
These results indicate that a-amvlase is the major various nmetabolic inllibitors
enzyme involved in degrading starci granules. Simi-
lar buit less extensive degradcation of the starch frac-
tions were observed dturinig the malting of barley sinmilar to but lecs dranmatic than tllose reported for
(12). 2 otlier pea varieties (29).
Starch Tn rnoz 'er in Cotyledonas of Germinating Chlorampilen,icol pretreatnlent produced clilorotic
P'eas. Labeling of the cotyledon oln the second to seedlinigs. This chemical lias previouslv been rie-
the sevenllth da of germination wvith a-D-glucose-U- ported to inliibit clhloroplast svnthlesis ( 21). Tlle
14C for 20 to 25 llr resulted in the incorporation of samples pretreated with nmg/nil of this reagent all
5 to 12 % of tile radioactivity taken up (7000-24,000 died wvitilin 9 days of germinationi, and only a few of
cl)m) into the starcli fraction. Practically the same those treated witil 2 nlg/nll were able to continue
resuilts wvere obtained witi sticro,e-U-14C. These growth. and produce greeni shoots by the thirteentil
r esuilts indicate tilat turnover of residuial starch mole- day. GroNvthi was very slow, in agreemeIlt with
ctules occurre(d even dulring the rapid pliase of starcll previous observations on sanlples growni in the dal-k
degradation. (35). Cvcloheximide at a concentratioll of 10 [g/nll
Effccts of Benzyladeninc anid Proteint Synthesis greatly retarded seedling growlth, starch degradation,
Inhibitors on1 Starch and Ainviase of Pca Cotylcdons. aIl(l a1-anivlase lproduliction (Fig. 3 ).
The effect of p)resoaking the see(i ill niletabolic inllibi-
tOIrs Oil starch aild amlIase lev'els of tile cotyledon Discussion
(h1ilring ger'milnatioln is shlownl ill Fig. 3. Zvimogranls
of these extracts sliowe(d sinlultaneous appearances During the first or 6 days of germination there
of the a-anlylase aild plhosphorvlase ( fast) bands. is a slow disappearance of starcil from the cotyle-
Growth, and starcli and enzyme levels of the thlird-dav dons. The rapid disappearance of starch which
saniples were the same as in the control. However, begins on the sixth day coincides with the appearance
later samples showed that p)retreatment witi 15 and of an a-amylase band and a second phosphorylase
30 Itg/nll chloramphenicol retarded growti, starcli band oin the gel electrophoresis zymograms. WVe
degradatioll, and enzyme production. Cyclolieximide conclude from these results that even though there
at a concentration of 2 jug/ml had no effect on is /8-amylase and phosphorylase activity (2 or 3
germination. bands and 1 band, respectively, on the zymograms)
Benzyladenin.e pretreatment caused thickening and in the dry cotyledons and throughout the period of
stunting of the axis and as many as 4 lateral shoots growth studied, these enzymes alone cannot bring
had grown at the cotyledonarv node by the seventh about a rapid degradation of starch. a-Amvlase,
day of germillation. The starcli and a-amvlase data and perlhaps the second phosphorvlase. is required
for tile samllplles pretreated withl benzyladenine wvere for rapid degradation of starch. Thle thiird (fast)
 JULIANO AND VAR-NER-STARCII DEGRADATION IN GER-MINATING PEAS 891
,8-amvlase band which appears during germiiination their sequence of production remained the same,
can also be produced by storing the extract of dry although their rates of production were reduced.
cotyledons for a few days at 0°. We conclude that Germination in the dark did not inhibit the p)roduc-
the fast fl-amylase band is present in a latent form tion of phosphorylase or a-amylase. Presumably, the
in the seed and becomes freed during germination. seqtuence of production of these 2 enzymes in the
A similar activation has been observed in wheat. dark is the same as in the light since Swain and
,8-Amylases whiclh are attached by disulfide bonds to Dekker (31) found peak phosphorylase activity at
reserve proteins are freed by reduction during ger- about tlle eighth day, whereas YounIg and Varner
mination and can be freed in vitro by treatment of (36) reported the specific activity of a-amylase as
homogenates with fl-mercaptoethanol (26). The still increasing 8 days after germination.
constant low total amylase activity during the first
few days must be due to 8-amnlyase, whiclh also
reduces the starch-iodine color at 620 m,u. Literature Cited
A similar, low activity of P-aylvase in pea coty-
ledons has been reported by Swain and Dekker (31)
throughout 8 to 10 days growth in the dark. Mlatile
(22) reported 8-anmylase activity of protein bodies 1. BAINN, J. MJ. AND F. V. NIERCER. 1966. Subcellular
from pea cotyledons isolated 1 and 3 days after organization of the cotyledon in germinating seeds
and seedlings of PisumBl sativumi L. Australian J.
germination. Biol. Sci. 19: 69-84.
Not only does the rapid increase of a-amylase 2. BRIONES, V. P., L. G. MAGBANUA, AND B. 0. JULI-
activity coincide with the faster rate of starch ANO. 1968. Changes in physiochemical properties
degradation but also the drop in intrinsic viscosity of of starch of developing rice grain. Cereal Chem.
the amylopectin with no apparent change in its outer 45: 351-57.
chain length is consistent only with a-anylolysis of 3. BROWN. A. P. AND J. L. WRAY. 1968. Correlate(I
its inner chains to produce lower mioleculal Nveight changes of some enzyme activities and cofactors
fragments with the same degree of branching. Phos- and substrate contents of pea cotyledons tissue
during germination. Biochem. J. 108: 437-44.
phorlase and f8-anvlase can only act. stepwise oni 4. CHRISPEELS, M. J. AND J. E. VARNER. 1967. Gib-
the outer chains of amvlopectin ('13, 17). The drop berellic acid-enhanced synthesis and release of a-
in viscosity of amivlose during this period of rapid amylase and ribonuclease by isolated barlcy alcu-
starch degradation also cannot be duie to 8-anvlvase rone layers. Plant Physiol. 42: 398-406.
aincd/or phosplhorylase actioni alonie as otherwise the 5. DANIELSSON, C. E. 195 1. The breakdo- n of the
,8-limit slhould decrease ratlher than increase. These high molecular reserve proteins of peas durinig
results indicate that a-amylase is the major enzyme germination. Acta Chem. Scand. 5: 541-54.
involved in the initial degradation of starch into the 6. DE FEKETE, M4. A. R. 1968. Die Rolle der Phos-
more soluble forms. wlhiclh other enzymes such as phorylase im Stoffwechsel der Starke in den Plas-
tiden. Planta 79: 208-21.
,6-amylase and phosphorylase assist in converting 7. FREDRICK, J. F. 1963. An algal a-glucainphospho-
into free sugars. This latter conversion of degraded rylase which required adenosiine-5-phosphate as
starch into dextrin and ultimately into free sugars, coenzyme. Phytochemistry 2: 413-15.
and subsequent translocation from the cotyledon must 8. FRYDENBERG! 0. AND G. NIELSEN. 1965. Am11ylase
be very efficient since no appreciable accunmutlation isozymes in germinatinig barley seeds. Hereditas
of dextrin or free sugars was observed. Presumably. 54: 123-39.
the low a-am-lase level is the reason for the slow 9. GATES, J. W. AND G. N4. Si.iPsox. 1968. The
presence of starch and alpha-amylase in the leaves
rate of starch degradation during the first 4 days of of plants. Can. J. Biochen. 46: 1459-62.
germination. The incorporation of a-D-glucose-U- 10. GILBERT, G. A. AND S. P. SPRAGG. 1964. Iodo-
'4C into the starch of 7-day seedling cotyledons metric determination of amylase. Iodine sorption:
showed turnover of the starch even during its rapid "Blue Value". Methods Carbohydrate Chem. 4:
degradation. In vitro studies have showni that only 168-69.
a-amylase can react onl starch grannules (34). How- 11. GREENW OOD, C. T. 1964. Viscosity - miiolecular
ever, the periods of activity of phosphorylase and weight relations. Mlolecular weight of amylase
amylases overlapped in this system, and complete and amylopectin by means of the limiting viscosity
separation of the periods of activity of these enzymes number. Methods Carbohydrate Chem. 4: 179-88.
12. GREENWN-OOD, C. T. AND J. THO'MSON. 1959. A
by blocking the production of either enzyme is re- comparison of the starches from barley and malted
quired to prove unequivocablv that a-amylase is the barley. J. Inst. Brewing 65: 346-53.
major enzyme for initial degradation of the starcl 13. GREENWN-OOD, C. T. AND J. THOxirsoN. 1962. Studies
granule. The leaves of 79 species of plants-all on the biosynthlesis of starch granules. 2. The
those studied-were recently reported to have ca-amiy- properties of the components of starches froni
lase activity (9). smooth and wrinkled peas during growth. Biochetmi.
Attempts were made to inhibit selectively the J. 82: 156-64.
14. GRIFFIN. H. L. AND Y. V. WEu. 1968. Isolation
production of either phosphorvlase or a-amylase in and characterization of the potato a-1,4-glucan-x-
the cotyledon of germinating pea by the use of 1,4-glucan 6-glucosyltransferase. Biochiemistry 7:
metabolic inhibitors. However, in all treatments 306-72.
892 PLANT PHYSIOLOGY

15. HASSII), W. Z. AND E. F. NEUFELD. 1964. Quanti- 25. ROBYT, J. AND D. FRENCH. 1964. Purificationi andl
tative deterrnination of starch in plant tissues. action pattern of an amylase from Bacilluts poly-
MIethods Carbohydrate Chem. 4: 33-36. lmuv.a. Arch. Biochem. Biophys. 104: 338-45.
16. LEE, Y. P. 1960. Potato phosphorylase. I. Puri- 26. ROWvSELL, E. V. AND L. J. GOAI). 1962. Latent :-
fication, physicochemical properties and catalytic amylase of wheat: its mode of attachment to glu-
activity. Biochim. Biophys. Acta 43: 18-24. tenin and its release. Biochem. J. 84: 73p-74p.
17. LIDDLE, A. M., D. J. MANNERS, AND A. WRIGHT. 27. SCANDALIOS, J. G. 1969. The genetic regulation
1961. Studies on carbohydrate-metabolizing en- of multiple molecular forms of enzymes in plants:
zymes. 6. The action of potato phosphorylase a review. Biochem. (;enetics 3: 37-79.
(P-enzyme) on starch-type polysaccharides. Bio- 28. SIEPMANN, R. AND H. STEGEMAN. 1967. Ellnzymi-
chem. J. 80: 30-09. electrophorese in Einschl uss-Polymerisatenl des
18. LOWRY, 0. H., N. J. ROSENBROUGH, A. L. FAIR, Acrylamids. A. Amvlasen, Phosphorylaseni. Z.
AND R. J. RANDALL. 1951. Protein measurement
Naturforsch. 226: 949-55.
with the folin phenol reagenlt. J. Biol. Chem. 193: 29. SPRENT. J. 1. 1968. Effects of henlzyladenlinie on
265-90. cotyledon metabolisnm and growth of peas. Planta
19 MCCREADY, R. M., J. (GTGGOZ, V'. SILVEIRA, AND 81: 80-87.
H. S. OWENS. 1950. Determination of starch 30. SWAIN. R. R. ANI) E. E. D)EKKER. 1966. Seed ger-
and amylase in vegetables. Anal. Chem. 22: 1156- mination studies. 1. Purification and properties
58.
of an a-amylase from the cotyledons of germinat-
20. MAcDoNALD, T. AND M. D. tTPADHYA. 1968. Car-
ing peas. Biochim. Biophys. Acta 122: 75-86.
bohydrase isozymes in corn and potato. Plant 31. SWAI N R. R. ANT) E. E. DEKKER. 1966. See(d
Physiol. 43: 8-16. germination studies. II. Pathways for starch
21. MARGULIES, M. M. 1962. Effect of chlorampheni- degradation in germinating pea seedlings. Biochim.
col in light dependent development of seedlings of Biophys. Acta 122: 87-100.
Phascoluts 7'udgaris var. Black Valentine, with par- 32 TSAI, C. Y. AND 0. E. NELSON. 1968. Pliosplhorv-
ticular reference to development of photosynthetic lase I and IS in maize endosperm. Plant Physiol.
activity. Planlt Physiol. 37: 473-80. 43: 103-12.
22. MATILE, P. 1968. Aleurone vacuoles as lysosomes. 33. VARNER, J. E.. T. V. BALCE, ANI) R. C. HUANG.
Z. Pflanzenphysiol. 58: 365-68. 1963. Senescenice of cotyledons of germinating
23 POTTER, A. L., V. SILEIRA, R. M. MCCREADIY, AND peas. Influence of axis tissue. Plant Physiol.
H. S. OWENS. 1953. Fractionation of starches 38: 89-92.
from smooth and wrinkled seeded peas. Molecu- 34. WALKFR. (G. J. ANT) P. M1. HIOPE. 1963. Thle actionl
lar weights end-group assays and iodine affinities of some a-amvlases on starch granules. Biochenm.
of the fractions. J. Am. Chem. Soc. 75: 1335-38. J. 86: 452-62.
24. RINIDERZKNECHT, H., P. WILDING, AND B. J. HAVER- 35 YOUNG. J. L. AND J. E. VARNER. 1959. Enzymle
BAcK. 1967. A new method for the determination
of a-amylase. Experientia 23: 805. synthesis in the cotyledons of germinatinlg seeds.
Arch. Biochim. Biophys. 84: 71-78.